Sản xuất de novo flavonoid naringenin trong Saccharomyces cerevisiae được kỹ thuật hóa

Microbial Cell Factories - Tập 11 Số 1 - 2012
Frank Koopman1, Jules Beekwilder2, Barbara Crimi1, Adèle van Houwelingen3, Robert D. Hall2, Dirk Bosch2, Antonius J. A. van Maris1, Jack T. Pronk2, Jean‐Marc Daran2
1Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands
2Platform for Green Synthetic Biology, P.O. Box 5057, 2600 GA, Delft, The Netherlands
3Plant Research International (PRI), P.O. Box 16, 6700 AA Wageningen, The Netherlands,

Tóm tắt

Tóm tắt Nền tảng

Flavonoid là một gia đình lớn các hợp chất trung gian chuyển hóa thứ cấp của thực vật, thể hiện nhiều loại hình tính chất chống oxy hóa và liên quan đến sức khỏe con người. Sản xuất flavonoid từ thực vật bị giới hạn bởi năng suất thấp và độ phức tạp của các flavonoid thu hồi. Do đó, để vượt qua những hạn chế này, việc kỹ thuật hóa chuyển hóa của các con đường cụ thể trong các hệ thống vi sinh vật đã được dự kiến để sản xuất lượng lớn một phân tử đơn.

Kết quả

Saccharomyces cerevisiae đã được kỹ thuật hóa để sản xuất flavonoid trung gian quan trọng, naringenin, chỉ từ glucose. Để thực hiện điều này, các gen sinh tổng hợp naringenin cụ thể từ Arabidopsis thaliana đã được chọn lựa thông qua phân tích biểu hiện so sánh và được đưa vào S. cerevisiae. Việc chỉ biểu hiện các gen này từ A. thaliana đã tạo ra naringenin ở nồng độ ngoài tế bào thấp (<5.5 μM). Để tối ưu hóa nồng độ naringenin, một chủng sinh dưỡng nấm men đã được phát triển. Việc tổng hợp các axit amin thơm đã được điều chỉnh bằng cách giảm thiểu sự ức chế hồi tiếp của 3-deoxy-d-arabinose-heptulosonate-7-phosphate synthase (Aro3, Aro4) và giảm thiểu sự hình thành sản phẩm phụ bằng cách loại bỏ phenylpyruvate decarboxylase (Aro10, Pdc5, Pdc6). Cùng với việc tăng số lượng bản sao của gen chalcone synthase và sự biểu hiện của một tyrosine ammonia lyase dị hợp, các sửa đổi này đã dẫn đến việc tăng nồng độ naringenin ngoài tế bào gấp 40 lần (đạt khoảng 200 μM) trong các văn hóa flask lắc với glucose. Trong các phản ứng batch được kiểm soát pH và có oxy, nồng độ naringenin ngoài tế bào vượt quá 400 μM đã đạt được.

Kết luận

Kết quả được báo cáo trong nghiên cứu này chứng minh rằng S. cerevisiae có khả năng sản xuất de novo naringenin bằng cách đồng biểu hiện các gen sản xuất naringenin từ A. thaliana và tối ưu hóa dòng chảy hướng tới con đường naringenin. Nấm men được kỹ thuật hóa làm chủ thể sản xuất naringenin cung cấp một khung chuyển hóa để sản xuất một loạt flavonoid và khám phá chức năng sinh học của chúng.

Từ khóa


Tài liệu tham khảo

Xu P, Ranganathan S, Fowler ZL, Maranas CD, Koffas MA: Genome-scale metabolic network modeling results in minimal interventions that cooperatively force carbon flux towards malonyl-CoA. Metab Eng. 2011, 13: 578-587. 10.1016/j.ymben.2011.06.008

Facchini PJ, Bohlmann J, Covello PS, De Luca V, Mahadevan R, Page JE, Ro DK, Sensen CW, Storms R, Martin VJ: Synthetic biosystems for the production of high-value plant metabolites. Trends Biotechnol. 2012, 30: 127-131. 10.1016/j.tibtech.2011.10.001

Xiao ZP, Peng ZY, Peng MJ, Yan WB, Ouyang YZ, Zhu HL: Flavonoids Health Benefits and Their Molecular Mechanism. Mini-Rev Med Chem. 2011, 11: 169-177. 10.2174/138955711794519546

Wedick NM, Pan A, Cassidy A, Rimm EB, Sampson L, Rosner B, Willett W, Hu FB, Sun Q, van Dam RM: Dietary flavonoid intakes and risk of type 2 diabetes in US men and women. Am J Clin Nutr. 2012, 95: 925-933. 10.3945/ajcn.111.028894

Knekt P, Kumpulainen J, Jarvinen R, Rissanen H, Heliovaara M, Reunanen A, Hakulinen T, Aromaa A: Flavonoid intake and risk of chronic diseases. Am J Clin Nutr. 2002, 76: 560-568.

Wang B, Zhang X: Inhibitory effects of Broccolini leaf flavonoids on human cancer cells. Scanning. 2012, 34: 1-5. 10.1002/sca.20278

McCullough ML, Peterson JJ, Patel R, Jacques PF, Shah R, Dwyer JT: Flavonoid intake and cardiovascular disease mortality in a prospective cohort of US adults. Am J Clin Nutr. 2012, 95: 454-464. 10.3945/ajcn.111.016634

Martin C, Butelli E, Petroni K, Tonelli C: How can research on plants contribute to promoting human health?. Plant Cell. 2011, 23: 1685-1699. 10.1105/tpc.111.083279

Zava DT, Duwe G: Estrogenic and antiproliferative properties of genistein and other flavonoids in human breast cancer cells in vitro. Nutr Cancer. 1997, 27: 31-40. 10.1080/01635589709514498

Greenwald P: Clinical trials in cancer prevention: current results and perspectives for the future. J Nutr. 2004, 134: 3507S-3512S.

Hou DX, Fujii M, Terahara N, Yoshimoto M: Molecular mechanisms behind the chemopreventive effects of anthocyanidins. J Biomed Biotechnol. 2004, 2004: 321-325. 10.1155/S1110724304403040

Allister EM, Borradaile NM, Edwards JY, Huff MW: Inhibition of microsomal triglyceride transfer protein expression and apolipoprotein B100 secretion by the citrus flavonoid naringenin and by insulin involves activation of the mitogen-activated protein kinase pathway in hepatocytes. Diabetes. 2005, 54: 1676-1683. 10.2337/diabetes.54.6.1676

Fowler ZL, Koffas MA: Biosynthesis and biotechnological production of flavanones: current state and perspectives. Appl Microbiol Biotechnol. 2009, 83: 799-808. 10.1007/s00253-009-2039-z

Wang Y, Chen S, Yu O: Metabolic engineering of flavonoids in plants and microorganisms. Appl Microbiol Biotechnol. 2011, 91: 949-956. 10.1007/s00253-011-3449-2

Nijveldt RJ, van Nood E, van Hoorn DE, Boelens PG, van Norren K, van Leeuwen PA: Flavonoids: a review of probable mechanisms of action and potential applications. Am J Clin Nutr. 2001, 74: 418-425.

Limem I, Guedonc E, Hehn A, Bourgaud F, Ghedira LC, Engasser JM, Ghoul M: Production of phenylpropanoid compounds by recombinant microorganisms expressing plant-specific biosynthesis genes. Process Biochem. 2008, 43: 463-479. 10.1016/j.procbio.2008.02.001. 10.1016/j.procbio.2008.02.001

Leonard E, Lim KH, Saw PN, Koffas MA: Engineering central metabolic pathways for high-level flavonoid production in Escherichia coli. Appl Environ Microbiol. 2007, 73: 3877-3886. 10.1128/AEM.00200-07

Santos CNS, Koffas M, Stephanopoulos G: Optimization of a heterologous pathway for the production of flavonoids from glucose. Metab Eng. 2011, 13: 392-400. 10.1016/j.ymben.2011.02.002

Trantas E, Panopoulos N, Ververidis F: Metabolic engineering of the complete pathway leading to heterologous biosynthesis of various flavonoids and stilbenoids in Saccharomyces cerevisiae. Metab Eng. 2009, 11: 355-366. 10.1016/j.ymben.2009.07.004

Watts KT, Lee PC, Schmidt-Dannert C: Exploring recombinant flavonoid biosynthesis in metabolically engineered Escherichia coli. Chem Biochem. 2004, 5: 500-507.

Koopmann E, Hahlbrock K: Differentially regulated NADPH: cytochrome P450 oxidoreductases in parsley. Proc Natl Acad Sci USA. 1997, 94: 14954-14959. 10.1073/pnas.94.26.14954

Winkel-Shirley B: Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol. 2001, 126: 485-493. 10.1104/pp.126.2.485

Costa MA, Bedgar DL, Moinuddin SG, Kim KW, Cardenas CL, Cochrane FC, Shockey JM, Helms GL, Amakura Y, Takahashi H, et al: Characterization in vitro and in vivo of the putative multigene 4-coumarate:CoA ligase network in Arabidopsis: syringyl lignin and sinapate/sinapyl alcohol derivative formation. Phytochem. 2005, 66: 2072-2091. 10.1016/j.phytochem.2005.06.022. 10.1016/j.phytochem.2005.06.022

Burbulis IE, Winkel-Shirley B: Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway. Proc Natl Acad Sci USA. 1999, 96: 12929-12934. 10.1073/pnas.96.22.12929

Winkel BSJ: Metabolic channeling in plants. Annu Rev Plant Biol. 2004, 55: 85-107. 10.1146/annurev.arplant.55.031903.141714

Yu O, Shi J, Hession AO, Maxwell CA, McGonigle B, Odell JT: Metabolic engineering to increase isoflavone biosynthesis in soybean seed. Phytochem. 2003, 63: 753-763. 10.1016/S0031-9422(03)00345-5. 10.1016/S0031-9422(03)00345-5

Jiang H, Wood KV, Morgan JA: Metabolic engineering of the phenylpropanoid pathway in Saccharomyces cerevisiae. Appl Environ Microbiol. 2005, 71: 2962-2969. 10.1128/AEM.71.6.2962-2969.2005

Yan Y, Chemler J, Huang L, Martens S, Koffas MA: Metabolic engineering of anthocyanin biosynthesis in Escherichia coli. Appl Environ Microbiol. 2005, 71: 3617-3623. 10.1128/AEM.71.7.3617-3623.2005

Gibson DG, Benders GA, Andrews-Pfannkoch C, Denisova EA, Baden-Tillson H, Zaveri J, Stockwell TB, Brownley A, Thomas DW, Algire MA, et al: Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science. 2008, 319: 1215-1220. 10.1126/science.1151721

Shao Z, Zhao H: DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res. 2009, 37: e16-, 10.1093/nar/gkn724

Jiang H, Morgan JA: Optimization of an in vivo plant P450 monooxygenase system in Saccharomyces cerevisiae. Biotechnol Bioeng. 2004, 85: 130-137. 10.1002/bit.10867

Zhang YH: Substrate channeling and enzyme complexes for biotechnological applications. Biotechnol Adv. 2011, 29: 715-725. 10.1016/j.biotechadv.2011.05.020

Raes J, Rohde A, Christensen JH, Van de Peer Y, Boerjan W: Genome-wide characterization of the lignification toolbox in Arabidopsis. Plant Physiol. 2003, 133: 1051-1071. 10.1104/pp.103.026484

Craigon DJ, James N, Okyere J, Higgins J, Jotham J, May S: NASCArrays: a repository for microarray data generated by NASC's transcriptomics service. Nucleic Acids Res. 2004, 32: D575-D577. 10.1093/nar/gkh133

Toufighi K, Brady SM, Austin R, Ly E, Provart NJ: The botany array resource: e-northerns, expression angling, and promoter analyses. Plant J. 2005, 43: 153-163. 10.1111/j.1365-313X.2005.02437.x

Saito K, Hirai MY, Yonekura-Sakakibara K: Decoding genes with coexpression networks and metabolomics - 'majority report by precogs'. Trends Plant Sci. 2008, 13: 36-43. 10.1016/j.tplants.2007.10.006

Ehlting J, Buttner D, Wang Q, Douglas CJ, Somssich IE, Kombrink E: Three 4-coumarate:coenzyme A ligases in Arabidopsis thaliana represent two evolutionarily divergent classes in angiosperms. Plant J. 1999, 19: 9-20. 10.1046/j.1365-313X.1999.00491.x

Schnappauf G, Hartmann M, Kunzler M, Braus GH: The two 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isoenzymes from Saccharomyces cerevisiae show different kinetic modes of inhibition. Arch Microbiol. 1998, 169: 517-524. 10.1007/s002030050605

Hartmann M, Schneider TR, Pfeil A, Heinrich G, Lipscomb WN, Braus GH: Evolution of feedback-inhibited beta/alpha barrel isoenzymes by gene duplication and a single mutation. Proc Natl Acad Sci USA. 2003, 100: 862-867. 10.1073/pnas.0337566100

Luttik MA, Vuralhan Z, Suir E, Braus GH, Pronk JT, Daran JM: Alleviation of feedback inhibition in Saccharomyces cerevisiae aromatic amino acid biosynthesis: quantification of metabolic impact. Metab Eng. 2008, 10: 141-153. 10.1016/j.ymben.2008.02.002

Vuralhan Z, Luttik MA, Tai SL, Boer VM, Morais MA, Schipper D, Almering MJ, Kotter P, Dickinson JR, Daran JM, Pronk JT: Physiological characterization of the ARO10-dependent, broad-substrate-specificity 2-oxo acid decarboxylase activity of Saccharomyces cerevisiae. Appl Environ Microbiol. 2005, 71: 3276-3284. 10.1128/AEM.71.6.3276-3284.2005

Vuralhan Z, Morais MA, Tai SL, Piper MD, Pronk JT: Identification and characterization of phenylpyruvate decarboxylase genes in Saccharomyces cerevisiae. Appl Environ Microbiol. 2003, 69: 4534-4541. 10.1128/AEM.69.8.4534-4541.2003

Hazelwood LA, Daran JM, van Maris AJ, Pronk JT, Dickinson JR: The Ehrlich pathway for fusel alcohol production: a century of research on Saccharomyces cerevisiae metabolism. Appl Environ Microbiol. 2008, 74: 2259-2266. 10.1128/AEM.02625-07

Romagnoli G, Luttik MA, Kotter P, Pronk JT, Daran JM: Substrate specificity of thiamine-pyrophosphate-dependent 2-oxo-acid decarboxylases in Saccharomyces cerevisiae. Appl Environ Microbiol. 78: 7538-7548.

Flikweert MT, de Swaaf M, van Dijken JP, Pronk JT: Growth requirements of pyruvate-decarboxylase-negative Saccharomyces cerevisiae. FEMS Microbiol Lett. 1999, 174: 73-79. 10.1111/j.1574-6968.1999.tb13551.x

Zuurbier KWM, Fung SY, Scheffer JJC, Verpoorte R: Assay of chalcon synthase activity by high-performance liquid-chromatography. Phytochem. 1993, 34: 1225-1229. 10.1016/0031-9422(91)80005-L. 10.1016/0031-9422(91)80005-L

Hwang EI, Kaneko M, Ohnishi Y, Horinouchi S: Production of plant-specific flavanones by Escherichia coli containing an artificial gene cluster. Appl Environ Microbiol. 2003, 69: 2699-2706. 10.1128/AEM.69.5.2699-2706.2003

Kyndt JA, Meyer TE, Cusanovich MA, Van Beeumen JJ: Characterization of a bacterial tyrosine ammonia lyase, a biosynthetic enzyme for the photoactive yellow protein. FEBS Lett. 2002, 512: 240-244. 10.1016/S0014-5793(02)02272-X

De Deken RH: The Crabtree effect: a regulatory system in yeast. J Gen Microbiol. 1966, 44: 149-156.

Van Hoek P, Van Dijken JP, Pronk JT: Effect of specific growth rate on fermentative capacity of baker's yeast. Appl Environ Microbiol. 1998, 64: 4226-4233.

van Maris AJA, Bakker BM, Brandt M, Boorsma A, de Mattos MJT, Grivell LA, Pronk JT, Blom J: Modulating the distribution of fluxes among respiration and fermentation by overexpression of HAP4 in Saccharomyces cerevisiae. FEMS Yeast Research. 2001, 1: 139-149.

Winkel-Shirley B: Evidence for enzyme complexes in the phenylpropanoid and flavonoid pathways. Physiol Plant. 1999, 107: 142-149. 10.1034/j.1399-3054.1999.100119.x. 10.1034/j.1399-3054.1999.100119.x

Dueber JE, Wu GC, Malmirchegini GR, Moon TS, Petzold CJ, Ullal AV, Prather KL, Keasling JD: Synthetic protein scaffolds provide modular control over metabolic flux. Nat Biotechnol. 2009, 27: 753-759. 10.1038/nbt.1557

Moon TS, Dueber JE, Shiue E, Prather KLJ: Use of modular, synthetic scaffolds for improved production of glucaric acid in engineered E. coli. Metab Eng. 2010, 12: 298-305. 10.1016/j.ymben.2010.01.003

Wang Y, Yu O: Synthetic scaffolds increased resveratrol biosynthesis in engineered yeast cells. J Biotechnol. 2011, 157: 258-260.

Kim IK, Roldao A, Siewers V, Nielsen J: A systems-level approach for metabolic engineering of yeast cell factories. FEMS Yeast Res. 2012, 12: 228-248. 10.1111/j.1567-1364.2011.00779.x

Santos CNS: Combinatorial search strategies for the metabolic engineering of microorganisms. 2010, Cambridge, MA: Massachusetts Institute of Technology, PhD thesis,2010.

Jez JM, Ferrer JL, Bowman ME, Dixon RA, Noel JP: Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase. Biochemistry. 2000, 39: 890-902. 10.1021/bi991489f

Beekwilder J, Wolswinkel R, Jonker H, Hall R, de Vos CH, Bovy A: Production of resveratrol in recombinant microorganisms. Appl Environ Microbiol. 2006, 72: 5670-5672. 10.1128/AEM.00609-06

Ruenwai R, Cheevadhanarak S, Laoteng K: Overexpression of acetyl-CoA carboxylase gene of Mucor rouxii enhanced fatty acid content in Hansenula polymorpha. Mol Biotechnol. 2009, 42: 327-332. 10.1007/s12033-009-9155-y

Shi S, Valle-Rodriguez JO, Khoomrung S, Siewers V, Nielsen J: Functional expression and characterization of five wax ester synthases in Saccharomyces cerevisiae and their utility for biodiesel production. Biotechnol Biofuels. 2012, 5: 7-, 10.1186/PREACCEPT-1932279820621895

Wattanachaisaereekul S, Lantz AE, Nielsen ML, Nielsen J: Production of the polyketide 6-MSA in yeast engineered for increased malonyl-CoA supply. Metab Eng. 2008, 10: 246-254. 10.1016/j.ymben.2008.04.005

Leonard E, Yan Y, Fowler ZL, Li Z, Lim CG, Lim KH, Koffas MA: Strain improvement of recombinant Escherichia coli for efficient production of plant flavonoids. Mol Pharm. 2008, 5: 257-265. 10.1021/mp7001472

Naesby M, Nielsen SV, Nielsen CA, Green T, Tange TO, Simon E, Knechtle P, Hansson A, Schwab MS, Titiz O, et al: Yeast artificial chromosomes employed for random assembly of biosynthetic pathways and production of diverse compounds in Saccharomyces cerevisiae. Microb Cell Fact. 2009, 8: 45-, 10.1186/1475-2859-8-45

van Dijken JP, Bauer J, Brambilla L, Duboc P, Francois JM, Gancedo C, Giuseppin ML, Heijnen JJ, Hoare M, Lange HC, et al: An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. Enzyme Microb Technol. 2000, 26: 706-714. 10.1016/S0141-0229(00)00162-9

Nijkamp JF, van den Broek M, Datema E, de Kok S, Bosman L, Luttik MA, Daran-Lapujade P, Vongsangnak W, Nielsen J, Heijne WH, et al: De novo sequencing, assembly and analysis of the genome of the laboratory strain Saccharomyces cerevisiae CEN.PK113–7D, a model for modern industrial biotechnology. Microb Cell Fact. 2012, 11: 36-, 10.1186/1475-2859-11-36

Verduyn C, Postma E, Scheffers WA, Vandijken JP: Physiology of Saccharomyces cerevisiae in Anaerobic Glucose-Limited Chemostat Cultures. J Gen Microbiol. 1990, 136: 395-403. 10.1099/00221287-136-3-395

Pronk JT: Auxotrophic yeast strains in fundamental and applied research. Appl Environ Microbiol. 2002, 68: 2095-2100. 10.1128/AEM.68.5.2095-2100.2002

Entian KD, Kotter P: 25 Yeast genetic strain and plasmid collections. Method Microbiol. 2007, 36: 629-666.

Gueldener U, Heinisch J, Koehler GJ, Voss D, Hegemann JH: A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic Acids Res. 2002, 30: e23-, 10.1093/nar/30.6.e23

Alberti S, Gitler AD, Lindquist S: A suite of Gateway (R) cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae. Yeast. 2007, 24: 913-919. 10.1002/yea.1502

Mumberg D, Muller R, Funk M: Yeast Vectors for the Controlled Expression of Heterologous Proteins in Different Genetic Backgrounds. Gene. 1995, 156: 119-122. 10.1016/0378-1119(95)00037-7

de Kok S, Yilmaz D, Suir E, Pronk JT, Daran JM, van Maris AJ: Increasing free-energy (ATP) conservation in maltose-grown Saccharomyces cerevisiae by expression of a heterologous maltose phosphorylase. Metab Eng. 2011, 13: 518-526. 10.1016/j.ymben.2011.06.001

Yamada K, Lim J, Dale JM, Chen H, Shinn P, Palm CJ, Southwick AM, Wu HC, Kim C, Nguyen M, et al: Empirical analysis of transcriptional activity in the Arabidopsis genome. Science. 2003, 302: 842-846. 10.1126/science.1088305

Seki M, Narusaka M, Kamiya A, Ishida J, Satou M, Sakurai T, Nakajima M, Enju A, Akiyama K, Oono Y, et al: Functional annotation of a full-length Arabidopsis cDNA collection. Science. 2002, 296: 141-145. 10.1126/science.1071006

Gietz RD, Schiestl RH: High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc. 2007, 2: 31-34. 10.1038/nprot.2007.13

Noskov VN, Koriabine M, Solomon G, Randolph M, Barrett JC, Leem SH, Stubbs L, Kouprina N, Larionov V: Defining the minimal length of sequence homology required for selective gene isolation by TAR cloning. Nucleic Acids Res. 2001, 29: E32-, 10.1093/nar/29.6.e32

Grote A, Hiller K, Scheer M, Munch R, Nortemann B, Hempel DC, Jahn D: JCat: a novel tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Res. 2005, 33: W526-W531. 10.1093/nar/gki376

Watts KT, Mijts BN, Lee PC, Manning AJ, Schmidt-Dannert C: Discovery of a substrate selectivity switch in tyrosine ammonia-lyase, a member of the aromatic amino acid lyase family. Chem Biol. 2006, 13: 1317-1326. 10.1016/j.chembiol.2006.10.008

De Vos RC, Moco S, Lommen A, Keurentjes JJ, Bino RJ, Hall RD: Untargeted large-scale plant metabolomics using liquid chromatography coupled to mass spectrometry. Nat Protoc. 2007, 2: 778-791. 10.1038/nprot.2007.95